Institute of Physiology - Project


ARRS N3-0133

Beta-cells in diet-induced diabetes and remission

Celice beta med razvojem in remisijo z dieto povzročene sladkorne bolezni

Coordinating person: prof. dr. Andraž Stožer (Faculty of Medicine University of Maribor)

Summary:
80 % of short-duration, and around 50 % of long-duration patients with T2DM can become normoglycaemic without medication (i.e., disease-free) after weight loss achieved with a very-low-calorie diet (VLCD) and around 60 % of short-duration and 20 % of long-duration diabetic patients are able to sustain normoglycemia by means of an isocaloric diet for at least 6 months thereafter. The observed improvement of glucose metabolism is accompanied by an acute reduction in liver fat content, an improved liver insulin sensitivity and reduced endogenous glucose output, as well as by a reduction in pancreas fat content and an improved beta-cell function, with a largely unchanged peripheral (i.e., adipose and muscular) insulin sensitivity.

Genetic and dietary mouse models of T2DM do not satisfactorily mirror the human situation and there are only a few studies studying the reversibility of the beta cell defect. Pathophysiological studies are based on models of beta cell function that involve dispersed beta cells or isolated islets and largely ignore beta cell functional coupling in multicellular syncytia, despite the growing evidence showing that cell–cell interactions are a prerequisite for a proper hormone secretion and despite the fact that the acute tissue slice methodological approach enables studying these aspects of beta cell physiology.

We will develop and validate a novel diet-induced mouse model of T2DM, i.e. the so called western diet (WD)-fed mice, with a higher translational value due to a diet that better mimics the composition of a western human diet and due to a dietary intervention to reverse the beta cell defect. Beta cell function will be studies in this model using the acute tissue slice approach in combination with electro-, optophysiological, as well as advanced graph-theoretical analytical methods.

Main emphasis of the project will be the mechanism of the reversal of T2DM following the intermittent fasting with VLCD, that results in normoglycemia in T2DM.


Figure 1: The methodological approach of the project. A C57BL/6J mice will be fed with western diet. Following metabolic derrangement, a subset will be exposed to a dietary intervention. B-J Methods used in the project. After tissue harvesting (F) we will apply patch-clamp (B), calcium and membrane potential optical recording (C), slow photolysis of caged calcium (D), graph-teoretical approaches (G), and (immuno)histological staining (J). In vivo measurements will include assessing levels of glucose, triglycerides and cholesterol (E), body weight (H) and activity patterns (I).

Koordinator projekta: izr. prof. dr. Andraž Stožer (Medicinska fakulteta Univerza v Mariboru)

Vsebina:
Več kot 80 % bolnikov s kratkotrajno SB2 in približno 50 % bolnikov z dolgotrajno SB2 doseže normoglikemijo brez dodatnega zdravljenja (t. j. ozdravitev) po izgubi telesne mase zaradi zelo nizkokalorične diete (VLCD, angl. very-low-calorie diet) in okrog 60 % bolnikov s kratkotrajno ter 20 % bolnikov z dolgotrajno SB2 lahko s pomočjo izokalorične diete vzdržuje normoglikemijo vsaj še 6 mesecev po VLCD. Opisano izboljšanje presnove glukoze spremljajo zmanjšanje vsebnosti maščob v jetrih, izboljšana občutljivost jeter na inzulin in zmanjšanje endogene proizvodnje glukoze, prav tako zmanjšanje vsebnosti maščob v trebušni slinavki in izboljšano delovanje celic beta, ob nespremenjeni periferni občutljivosti na inzulin (v maščobnem in skeletnomišičnem tkivu). 

Genetski in prehranski modeli SB2 neustrezno odražajo razvoj SB2 pri človeku in le nekaj raziskav proučuje reverzibilnost okvar celic beta [24, 25]. Patofiziološke raziskave imajo za osnovo modele funkcije celic beta, ki vključujejo razpršene celice beta ali izolirane otočke in večinoma ignorirajo funkcionalno sklopljenost celic beta v večceličnem sinciciju, kljub naraščajočim dokazom, da so medcelične povezave predpogoj za normalno izločanje hormonov in kljub dejstvu, da metodološki pristop z akutno tkivno rezino omogoča študij teh vidikov fiziologije celic beta [26-30]. 

Razvili in validirali bomo nov, s prehrano povzročenega modela SB2, t. i. miši, hranjenih z zahodnjaško hrano (WD, angl. western diet), ki ima boljšo translacijsko vrednost zaradi dejstva, da je ta hrana bolj primerljiva zahodnjaški prehrani po svoji sestavi in zaradi diete za namene poprave poškodb funkcije celic beta. Funkcijo celic beta v tem modelu bomo študirali z uporabo priprave akutnih tkivnih rezin v povezavi z elektro- in optofiziološkimi metodami kot tudi z analitičnimi metodami grafično-teoretičnega pristopa. 

Glavni poudarek bo raziskovanje popravljalnih mehanizmov po kratkotrajnem stradanju z VLCD, ki omogočijo normoglikemijo pri SB2.


Slika 1: Metodološki pristop. A Miši C57BL/6J bomo hranili z WD. Po nastopu metabolne motnje bomo del miši izpostavili prehranski intervenciji. B-J Metode uporabljene v projektu. Po pridobitvi tkiv (F) bomo uporabili metodo vpete-krpice membrane (B), optične meritve kalcija in membranskega potenciala (C), počasno sproščanje ujetega kalcija (D), grafično-teoretični pristop (G) in (imuno)histološko barvanje (J). Meritve in vivo bodo vključevale določevanja nivoja glukoze, trigliceridov in holesterola (E), telesne teže (H) in vzorca aktivnosti (I).
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